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Digestive phospholipase A(2) in insects
Abstract
We hypothesized that phospholipase A2 (PLA2) is a common feature of insect digestive physiology in insects. PLA2 hydrolyzes polyunsaturated fatty acids (PUFAs) associated with the sn-2 position of phospholipids (PLs). We conducted experiments to determine whether insects produce a digestive PLA2, and investigated its biochemical and physiological characteristics. Oral secretions of adult burying beetles, Nicrophorus marginatus , display a calcium-dependent PLA2 similar to its mammalian counterparts. However, PLA2 activity in midgut contents of the tobacco hornworm larvae, Manduca sexta, is independent of calcium. PLA2 activity in both insects is sensitive to reaction pH, protein concentration, substrate concentration, temperature, and incubation time. We also found that fed hornworms expressed higher PLA2 activity than starved ones. Based on in vitro assay, midguts isolated from M. sexta are competent to secrete PLA2 into the incubation medium. Two-hour incubations with buffer amended with selected diet component resulted in increased secretion of digestive PLA 2, while small increases in secretion were detected when the buffer was amended with a specific PL, phosphatidylcholine. Highest secretion of the enzyme was detected from the middle region and lowest secretion from the anterior midgut region. Because isolated midguts responded to food chemicals with increased secretion of digestive PLA2, we suggest the secretion is regulated by a prandial and/or paracrine mechanism, as suggested for digestive proteases in other insect species. Our efforts to partially purify the digestive PLA2 yielded three interesting points. First, upon elution from superose-12 size-exclusion column, fractions containing the highest enzyme activity was determined to be about 24 kDa, which is higher than the mammalian secretory PLA2. Second, in the absence of ammonium sulfate precipitation, we obtained more than 100% recovery of enzyme activity, which is equivalent to a 1.5-fold purification of the enzyme. And third, three major proteins were displayed upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the most active fraction. One of which was identified to be trypsin (43 kDa), based on its similarities with trypsins identified from Manduca.
Subject Area
Entomology|Biochemistry
Recommended Citation
Rana, Rico Lamoste, "Digestive phospholipase A(2) in insects" (1999). ETD collection for University of Nebraska-Lincoln. AAI9942148.
https://digitalcommons.unl.edu/dissertations/AAI9942148